Methods for treatment of vascular endothelial dysfunction using nicotinamide mononucleotide

ABSTRACT

This invention is about the compositions and methods for assessing and treating vascular endothelial dysfunction. Various aspects provide a method for treatment of vascular endothelial dysfunction, comprising administering a composition comprising nicotinamide mononucleotide and a pharmaceutical excipient to a subject. In one embodiment the dose is administered to subjects in response to the indicator. In another embodiment the dose is administered chronically to subjects.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage patent application that claimspriority to, and the benefit of, PCT Patent Application PCT/US 15/22267,filed on Mar. 24, 2015, entitled “METHODS FOR TREATMENT OF VASCULARENDOTHELIAL DYSFUNCTION USING NICOTINAMIDE MONONUCLEOTIDE,” which claimspriority to U.S. Provisional Patent Application Ser. No. 61/969,658,filed Mar. 24, 2014, entitled “METHODS FOR TREATMENT OF VASCULARENDOTHELIAL DYSFUNCTION USING NICOTINAMIDE MONONUCLEOTIDE,” which areincorporated herein by reference in their entireties.

GOVERNMENT RIGHTS

This invention was made with government support under grant numbers R37AG013038 and AG000279 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF INVENTION

The present disclosure relates, generally, to compositions and methodsfor the treatment of vascular endothelial dysfunction using compositionscomprising nicotinamide mononucleotide.

BACKGROUND OF THE INVENTION

Cardiovascular diseases (CVD) remain the leading cause of death inmodern society in developed nations. The number of older adults in thedeveloped world is expected to at least double by 2050, and this isassociated with projections of marked increases in CVD burden.

Aging increases the risk of CVD largely due to the dysfunction of thearteries, namely endothelial dysfunction and large elastic arterystiffness. Vascular endothelial dysfunction is primarily assessed viaendothelium-dependent dilation (EDD) and is impaired largely due toincreased superoxide (O₂ ⁻) production. Increased O₂ ⁻ reduces thebioavailability of the potent vasodilator and vasoprotective moleculenitric oxide (NO). Increased aortic stiffness, in particular, reducesthe ability to buffer increases in pressure produced by systolicejection of blood into the large elastic arteries with each cardiaccontraction. This increases systolic blood pressure and arterial pulsepressure (the difference between systolic and diastolic blood pressure),as well as the “pulsatility” of blood flow, which is transmitted to themicrovasculature of vulnerable high-flow organs such as the brain andkidney, causing end-organ damage and other pathophysiological effects.Similarly, increased arterial stiffness has been linked to endothelialdysfunction and is now recognized as the major independent risk factorfor age-associated CVD. Therefore, there is an urgent need to developtreatments that reduce the risk of CVD with aging.

Without being bound by theory, the mechanisms by which arteries stiffenwith age are not completely understood, but are thought to includechanges in the composition of structural proteins within the arterialwall. Collagen (type I) is the primary load-bearing protein in thearterial wall, and its abundance is increased with advancing age. Incontrast, elastin, the main structural protein conferring elasticity, isreduced in old arteries. It has previously been shown in mice (FleenorB. S. et al. 2012a, Aging Cell. 11, 269-276) and cultured aorticfibroblasts (Fleenor B. S. et al. 2010, J Physiol. 588, 3971-3982) thatoxidative stress contributes to some or all of the age-associatedstructural changes seen within the arteries.

Two key antecedents and independent predictors of clinical CVD in olderadults are thought to include vascular endothelial dysfunction, assessedby endothelium-dependent dilation (EDD), and large elastic arterystiffness, measured by aortic pulse wave velocity (aPWV). A commonmechanism that contributes to both vascular endothelial dysfunction andlarge elastic artery stiffness with aging is believed to involveexcessive superoxide-associated vascular oxidative stress (Seals D. R.et al. 2011, Clin Sci 120, 357-375; Fleenor B. S. et al. 2012a, AgingCell. 11, 269-276; Bachschmid M. M. et al. 2013, Ann Med. 45, 17-36).Increased vascular production of superoxide occurs with aging andreduces the bioavailability of the vasoprotective and vasodilatorymolecule nitric oxide (NO), while also causing alterations in majorstructural proteins (collagen and elastin) in the large elastic arteries(i.e., the aorta and carotid arteries). These changes contributedirectly to age-related endothelial dysfunction and increased arterialstiffness. As such, treatments that reduce the excessive superoxideproduction in aging arteries hold the potential for improvingage-associated vascular dysfunction.

It is recognized that there is an association between endothelialdysfunction and a decline in cognitive and motor (physical) functionduring both normal aging and in age-associated disease states. It isfurther recognized that endothelial function plays a role in thesystemic regulation of metabolism, blood fluidity, tissue perfusion,immune function and enhancement of longevity.

It has been previously shown that lifelong caloric restriction (CR), aswell as short-term CR in old animals, prevents or reverses endothelialdysfunction and large elastic artery stiffening by reducing superoxideproduction, increasing NO bioavailability and modifying structuralproteins (Rippe C. et al. 2010 Aging Cell. 9, 304-312; Donato A. J. etal. 2013, Aging Cell. 12, 772-783). However, because adherence to CR isnot practical for most humans, there is growing interest inpharmacological therapies that may induce the benefits of CR.

Sirtuins are a class of enzyme proteins that possess deacylase activity,including deacetylase, desuccinylase, demalonylase, demyristoylase anddepalmitoylase activity, some of the sirtuins (for example, SIRT6) alsopossess mono-ribosyltransferase activity. The expression and activity ofsirtuin enzymes is reduced with advancing age. There are 7 mammaliansirtuins (SIRT 1-7) that correspond to the yeast Sir2 (silentmating-type information regulation). Sirtuins also possess nicotinamideadenine dinucleotide (NAD⁺)-dependent deacetylase activity. MammalianSIRT1, one of seven members in the sirtuin family of proteindeacetylases/deacylases, is a nicotinamide adenine dinucleotide(NAD⁺)-dependent deacetylase that acts as a metabolic energy sensorimplicated in several of the beneficial effects of CR, including reducedoxidative stress (Boily G. et al. 2008, PLoS One. 3, e1759; Merksamer P.I. et al. 2013, Aging (Albany N.Y.). 5, 144-150). Enhancing NAD⁺biosynthesis with NAD⁺ precursors, such as NMN and nicotinamide riboside(NR), increases the activity of the NAD⁺-dependent deacetylase SIRT1(Imai S. 2010, Pharmacol Res. 62, 42-47; Satoh A. et al. 2011, Handb ExpPharmacol. 206, 125-162; Canto C. et al. 2012, Cell Metab. 15, 838-847).

With advancing age, there is a reduction of the expression and activityof sirtuins in mammals and humans. Because of this, sirtuins arebelieved to influence many aging processes. SIRT1 expression inendothelial cells is positively associated with EDD in young and olderadults (Donato A. J. et al., 2011 J. Physiol. 589, 4545-4554), implyingthat SIRT1 may influence vascular function in humans. Previous studiesshow that reduced SIRT1 expression and activity is a key mechanismmediating impaired EDD in aging arteries (Rippe C. et al. 2010, AgingCell. 9, 304-312; Donato A. J. et al. 2011, J Physiol. 589, 4545-4554;Gano L. B. et al. 2014, Am J Physiol Heart Circ Physiol. 307,H1754-1763), and recent findings indicate that pharmacologicalactivation of SIRT1 with the compound SRT1720 improves EDD in old micein part by reducing oxidative stress (Gano L. B. et al. 2014, Am JPhysiol Heart Circ Physiol. 307, H1754-1763). Oxidative stress has beenshown to play an important role in the development of vascularendothelial dysfunction and large elastic artery stiffness associatedwith increasing age (Lakatta E. G. & Levy, 2003 Heart Fail Rev. 7,29-49; Seals et al. 2011, Clin Sci 120, 357-375). Oxidative stress inthe vasculature leads to a decrease in NO bioavailability, thus causingendothelial dysfunction and stiffening of the large elastic arteries.Superoxide reacts with NO, forming peroxynitrite (ONOO⁻), which reducesthe bioavailability of NO; this results in less bioavailable NO tocontribute to vasodilation. Furthermore, ONOO⁻ oxidizes tyrosineresidues on proteins post-translationally producing nitrotyrosine, onekey marker of oxidative stress.

It has been established that oxidative stress and inflammation areintimately connected (Csiszar A. et al. 2008, J Appl Physiol. 105,1333-1341; Ungvari Z. et al. 2010, J Gerontol A Biol Sci Med Sci. 65,1028-1041) and SIRT1 has been found to modulate the activity of nuclearfactor kappa B (NFκB) and tumor necrosis factor alpha (TNFα) (Yoshino Jet al., 2011, Cell Metab. 14, 528-536), both of which are masterregulators of the inflammatory process. The p65 subunit of NFκB is amajor target of SIRT1, and is deacetylated in response to SIRT1activation.

NAD⁺ bioavailability also decreases with age in various mammaliantissues, and restoring NAD⁺ levels has been shown to ameliorate high-fatdiet- and age-induced Type 2 Diabetes in mice while restoring geneexpression related to oxidative stress and inflammation to that of ahealthy, non-diabetic mouse, partly through SIRT1 activation.

SUMMARY OF THE INVENTION

Compositions and methods for assessing and treating vascular endothelialdysfunction are described herein. In various aspects, methods oftreating vascular endothelial dysfunction are provided. In variousembodiments, the method comprises determining an indicator of vascularendothelial dysfunction in a subject. In further embodiments, the methodcomprises administering a daily dose of a composition comprisingnicotinamide mononucleotide and a pharmaceutical excipient. In furtherembodiments, the dose of nicotinamide mononucleotide is from about 1 mgto about 25 mg per kg body weight per day. In further embodiments, thedose is from about 18 mg of nicotinamide mononucleotide per kg bodyweight per day. In further embodiments, the dose is administeredchronically to subjects. In further embodiments, the dose isadministered to subjects in response to the indicator.

In further embodiments, the methods comprise determining a subsequenteffect on vascular endothelial dysfunction in the subject. In furtherembodiments, the indicator of vascular endothelial dysfunction comprisesdetermining the extent of endothelium-dependent dilation and/or arterystiffness in a subject. In further embodiments, the extent ofendothelium-dependent dilation is associated with increased superoxideproduction. In further embodiments, the extent of endothelium-dependentdilation is associated with decreased SIRT1 expression. In furtherembodiments, the extent of endothelium-dependent dilation and/or arterystiffness is decreased in response to administration of the compositioncomprising nicotinamide mononucleotide. In further embodiments, thedecrease in the extent of endothelium-dependent dilation furthercomprises a decrease in superoxide production and an increase in nitricoxide bioavailability. In further embodiments, the decrease in theextent of endothelium-dependent dilation further comprises an increasein SIRT1 protein expression and activity. In further embodiments, thecomposition is administered over a period of time of about 30 days,about 3 months, about 6 months, about 12 months, about 18 months, about2 years, about 5 years, about 7 years, about 10 years, about 15 years,about 20 years, about 25 years, about 30 years, about 35 years, about 40years, or continued therapy over the lifetime of the subject.

In various aspects of the invention, NMN treatment selectively reducesstiffness in old animals. In further aspects, the methods of decreasingendothelium-dependent dilation and/or arterial stiffness in a subject,comprise determining the extent of endothelium-dependent dilation and/orarterial stiffness in a subject and administering a daily dose of acomposition comprising nicotinamide mononucleotide and a pharmaceuticalexcipient wherein the composition comprises from about 1 mg to about 25mg per kg body weight per day. In further embodiments, the dose is fromabout 18 mg of nicotinamide mononucleotide per kg body weight per day.In further embodiments, the dose is administered chronically to saidsubject. In further embodiments, a decrease in endothelium-dependentdilation and/or arterial stiffness is associated with an increase inbioavailability of nicotinamide adenine dinucleotide (NAD⁺). In furtherembodiments, a decrease in endothelium-dependent dilation and/orarterial stiffness further comprises a decrease in superoxideproduction, an increase in bioavailability of nitric oxide, and/or anincrease in SIRT1 protein expression and activity.

In aspects of the invention, NMN treatment activates SIRT1 and reducesinflammation and oxidative stress, resulting in improved vascularfunction.

In aspects of the invention, treatment with NMN reverses age-associatedvascular dysfunction by improving endothelial function and reducinglarge elastic artery stiffness in old C57Bl/6 mice, while restoring theage-related decline in SIRT1 protein expression and reducing oxidativestress. In aspects of the invention, treatment with NMN selectivelyrestores the activity of SIRT1 in the arteries of old mice to that ofyoung controls. In further aspects, the ratio of acetylated to total p65subunit of the transcription factor NFκB is decreased with NMNtreatment.

In aspects of the invention, NMN reverses large elastic arterystiffening associated with aging. In another aspect, NMN treatmentnormalizes collagen. In a further aspect, NMN treatment partiallypreserves elastin in the arterial wall.

Further aspects and embodiments will become apparent from the detaileddescription provided herein. It should be understood that thedescription and specific examples are intended for purposes ofillustration only and are not intended to limit the scope of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter of the present invention is particularly pointed outand distinctly claimed in the concluding portion of the specification. Amore complete understanding of the present invention, however, is bestobtained by referring to the detailed description and claims whenconsidered in connection with the drawing figures, wherein like numeralsdenote like elements and wherein:

FIG. 1 illustrates dose-responses to the endothelium-dependent dilatoracetylcholine (ACh) in young and old control (YC and OC) (FIG. 1A); doseresponses to young and old NMN-treated (YNMN and ONMN) mice NO-dependentdilation (FIG. 1B); and the endothelium-independent dilator sodiumnitroprusside (SNP) (FIG. 1C).

FIG. 2 illustrates maximal dose-response to the endothelium-dependentdilator acetylcholine (ACh) in young and old control (YC and OC) andyoung and old NMN-treated (YNMN and ONMN) mice in the presence orabsence of TEMPOL (FIG. 2A); superoxide production assessed by electronparamagnetic resonance (EPR) (FIG. 2B); and nitrotyrosine (NT) abundancein aorta (FIG. 2C).

FIG. 3 illustrates aortic pulse wave velocity (aPWV) in young and oldcontrol (YC and OC) and young and old NMN-treated (YNMN and ONMN) mice(FIG. 3A); elastic modulus (FIG. 3B); total arterial collagen-I (Col1)expression in aorta (FIG. 3C); and total elastin expression in aorta(FIG. 3D).

FIG. 4 illustrates SIRT1 expression in aorta of young and old control(YC and OC) and young and old NMN-treated (YNMN and ONMN) mice (FIG. 4A)and ratio of acetylated to total NFκB in aorta (FIG. 4B).

DETAILED DESCRIPTION

The following description is merely exemplary in nature and is notintended to limit the present invention, its applications, or its uses.It should be understood that throughout the drawings, correspondingreference numerals indicate like or corresponding parts and features.The description of specific examples indicated in various embodiments ofthe present invention are intended for purposes of illustration only andare not intended to limit the scope of the invention disclosed herein.Moreover, recitation of multiple embodiments having stated features isnot intended to exclude other embodiments having additional features orother embodiments incorporating different combinations of the statedfeatures.

Furthermore, the detailed description of various embodiments hereinmakes reference to the accompanying drawing figures, which show variousembodiments by way of illustration. While the embodiments are describedin sufficient detail to enable those skilled in the art to practice theinvention, it should be understood that other embodiments may berealized and that logical and mechanical changes may be made withoutdeparting from the spirit and scope of the present invention. Thus, thedetailed description herein is presented for purposes of illustrationonly and not of limitation. For example, steps or functions recited indescriptions of any method, system, or process, may be executed in anyorder and are not limited to the order presented. Moreover, any of thestep or functions thereof may be outsourced to or performed by one ormore third parties. Furthermore, any reference to singular includesplural embodiments, and any reference to more than one component mayinclude a singular embodiment.

As used herein, a “pharmaceutically acceptable excipient” refers to anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and the likethat are physiologically compatible. Some examples of pharmaceuticallyacceptable excipients include water, saline, phosphate buffered saline,dextrose, glycerol, ethanol and the like, as well as combinationsthereof. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol, sorbitol, orsodium chloride in the composition. Additional examples ofpharmaceutically acceptable excipients include wetting agents or minoramounts of auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the composition. Pharmaceutical compositions of the present inventionand methods for their preparation will be readily apparent to thoseskilled in the art. Such compositions and methods for their preparationmay be found, for example, in Remington's Pharmaceutical Sciences, 19thEdition (Mack Publishing Company, 1995). Pharmaceutical compositions arepreferably manufactured under GMP conditions. A pharmaceuticalcomposition of the invention may be prepared, packaged, or sold in bulk,as a single unit dose, or as a plurality of single unit doses. As usedherein, a “unit dose” is a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.In some embodiments, one or more active ingredients may be present inthe composition in addition to nicotinamide mononucleotide (NMN). Theamount of the active ingredient is generally equal to the dosage of theactive ingredient which would be administered to a subject or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

As used herein, a “therapeutically effective amount” or “effectiveamount” of a composition refers to an amount effective in the preventionor treatment of a disorder for the treatment of which the composition iseffective. A “disorder” refers to any condition that would benefit fromtreatment with the composition. In some embodiments, a composition ofthe invention is effective in the treatment of cardiovascular disease.In further embodiments, a composition of the invention is effective inthe treatment of vascular endothelial dysfunction.

As used herein, “treated,” “treating” or “treatment” refers to thediminishment or alleviation of at least one symptom associated or causedby the state, disorder or disease being treated. For example, treatmentcan be diminishment of one or more symptoms of a disorder or completeeradication of a disorder.

As used herein, “conditions,” “diseases” and “disorders” refers to andincludes aging, cardiovascular disease (CVD), atherosclerosis andendothelial dysfunction. In embodiments, the disorder may be a disorderassociated with aging, cardiovascular disease, atherosclerosis orendothelial dysfunction, for example cognitive impairments, Alzheimer'sDisease, motor dysfunction, insulin resistance and sarcopenia.

In various aspects, without being bound to any theory, NAD⁺ levels areincreased in old C57Bl/6 mice by administering the direct intracellularprecursor, nicotinamide mononucleotide (NMN), to increase sirtuinactivity, decrease oxidative stress and restore NO bioavailability.

In various aspects of the invention, nicotinamide mononucleotide (NMN)may be administered as an active ingredient in therapeutic compositions,for treating vascular endothelial dysfunction, among others. Generally,NMN is suitable to be administered in association with one or morepharmaceutically acceptable excipient(s). The term ‘excipient’ is usedherein to describe any ingredient other than the active ingredient. Thechoice of excipient(s) will to a large extent depend on factors such asthe particular mode of administration, the effect of the excipient onsolubility and stability, and the nature of the dosage form.

Actual dosage levels of the active ingredient(s) (for example, NMN) inpharmaceutical compositions and formulations may be varied so as toobtain an amount of the active ingredient that is effective to achievethe desired therapeutic response for a particular patient, composition,and mode of administration, without being toxic to the patient.

Without being bound to any theory, decreased NAD⁺ bioavailability isbelieved to contribute to age-associated vascular dysfunction. Invarious aspects, NMN may be administered to a subject in atherapeutically effective amount. In further aspects, endothelialfunction may be assessed via endothelium-dependent dilation (EDD), andaortic stiffness. Endothelial function may be assessed pre- andpost-treatment with NMN.

The selected dosage level will depend upon a variety of factorsincluding the activity of the composition found in the formulation, theroute of administration, the time of administration, the rate ofexcretion of the particular composition being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular composition employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

A physician having ordinary skill in the art can readily determine andprescribe the effective amount of the pharmaceutical composition of thepresent invention required. For example, the physician could start dosesof the composition of the invention employed in the pharmaceuticalformulation at levels lower than that required in order to achieve thedesired therapeutic effect and gradually increase the dosage until thedesired effect is achieved.

In various embodiments, the concentration of the active ingredient isbetween about 10 mg to about 6000 mg of nictotinamide mononucleotide perml of liquid formulation. In embodiments, the concentration of NMN isfrom about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250mg, about 300 mg, about 500 mg, about 750 mg, about 1000 mg, about 1200mg, about 1500 mg, about 2000 mg, about 2500 mg, about 3000 mg, about3500 mg, about 4000 mg, about 4500 mg, about 5000 mg, about 5500 mg, orabout 6000 mg per ml of liquid formulation. In embodiments, theconcentration of NMN may be calculated based on a subject's body weight.In embodiments, the concentration of NMN is between about 0.1 mg toabout 50 mg per kg body weight. In embodiments, the concentration of NMNis from about 0.1 mg to about 25 mg per kg body weight. In embodiments,the concentration of NMN is from about 1 mg, about 2.5 mg, about 5 mg,about 7.5 mg, about 10 mg, about 12.5 mg, about 15 mg, about 18 mg,about 20 mg, about 22.5 mg, or about 25 mg per kg body weight. Inembodiments, the concentration of NMN is from about 1 mg to about 25 mgper kg body weight. In embodiments, the concentration of NMN is fromabout 18 mg of nicotinamide mononucleotide per kg body weight per day.In embodiments, the concentration of NMN administered to a subject isfrom about 1,200 mg/day for a subject weighing about 150 lbs.

In various embodiments, a composition of the invention may beadministered as a daily dose over a period of time to a subject. Inembodiments, a composition of the invention may be administeredchronically or long-term. In embodiments, the composition may beadministered for a period of days, weeks, months, years or continuedtherapy over the lifetime of a subject. In embodiments, the compositionmay be administered for a period of about 30 days, about 3 months, about6 months, about 12 months, about 18 months, about 2 years, about 5years, about 7 years, about 10 years, about 15 years, about 20 years,about 25 years, about 30 years, about 35 years, or about 40 years. Inembodiments, a treatment regime may be determined for an individualsubject dependent on various factors. In some embodiments, a factor mayinclude, but not be limited to, a determination of the change in theextent of endothelial-dependent dilation and/or arterial stiffness inresponse to administration of the composition of the invention. Infurther embodiments, stiffening of the large elastic arteries withadvancing age can lead to an increase in arterial systolic and pulsepressures, left ventricular hypertrophy, and tissue damage to high-flowvital organs, such as the brain and kidneys. In embodiments, a subjectexhibiting an immediate response to the composition, for example, animmediate reduction in endothelial-dependent dilation and/or arterialstiffness, may require less frequent doses than a subject exhibiting aresponse to the composition at a later time or after several doses.

Any method for administering pharmaceutical or nutriceutical-likecompounds in the art may suitably be employed in accordance with theinvention.

Example

Supplementation of nicotinamide mononucleotide (NMN), a key NAD⁺intermediate, as potentially activating the mammalian NAD⁺-dependentdeacetylase SIRT1 and reverses age-associated vascular dysfunction andoxidative stress was tested. Old control mice (OC) had impaired carotidartery endothelium-dependent dilation (EDD) (60±5 vs. 84±2%), a measureof endothelial function, and nitric oxide (NO)-mediated EDD (37±4 vs.66±6%), compared with young mice (YC). This age-associated impairment inEDD was restored in OC by the superoxide (O₂ ⁻) scavenger TEMPOL(4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl; 82±7%). OC also hadincreased aortic pulse wave velocity (aPWV, 464±31 vs. 337±3 cm/s) andelastic modulus (EM, 6407±876 vs. 3119±471 kPa), measures of largeelastic artery stiffness, compared to YC. OC had greater aortic O₂ ⁻production (2.0±0.1 vs. 1.0±0.1 AU), nitrotyrosine abundance (a markerof oxidative stress), and collagen-I, and reduced elastin and vascularSIRT1 activity, measured by the acetylation status of the p65 subunit ofNFκB, compared to YC. Treatment with NMN in old mice restored EDD(86±2%) and NO-mediated EDD (61±5%), reduced aPWV (359±14 cm/s) and EM(3694±315 kPa), normalized O₂ ⁻ production (0.9±0.1 AU), decreasednitrotyrosine, reversed collagen-I, partially increased elastin, andrestored vascular SIRT1 activity. NMN treatment restores SIRT1 activityand reverses age-related arterial dysfunction and reduces oxidativestress.

Materials and Methods

Animals.

Young (4-8 months) C57Bl/6 male mice were purchased from Charles Riverand old (26-28 months) C57Bl/6 male mice were obtained from the NationalInstitute on Aging rodent colony. Mice were fed normal rodent chow adlibitum for the duration of the study. After an acclimation period of 2weeks, the young and old mice were divided into two subgroups: controlanimals (YC, OC) continued on normal drinking water and the otheranimals (YNMN, ONMN) received nicotinamide mononucleotide (NMN;Sigma-Aldrich Corp., St. Louis, Mo., USA) supplementation in thedrinking water (240 mg/kg/day) for 8 weeks. All mice were housed in ananimal care facility at the University of Colorado Boulder on a 12:12hour light-dark cycle. All animal procedures conformed to the Guide tothe Care and Use of Laboratory Animals (NIH publication No. 85-23,revised 1996) and were approved by the UCB Animal Care and UseCommittee.

Ex Vivo Carotid Artery Vasodilatory Responses.

EDD and endothelium-independent dilation were determined ex vivo inisolated carotid arteries as previously described (Rippe C. et al. 2010,Aging Cell. 9, 304-312; Sindler A. L. et al. 2011, Aging Cell. 10,429-437). Mice were anesthetized using isoflurane and euthanized byexsanguination via cardiac puncture. The carotid arteries were carefullyexcised, cannulated onto glass micropipette tips, and secured with nylon(11-0) sutures in individual myograph chambers (DMT Inc., Ann Arbor,Mich., USA) containing buffered physiological saline solutions. Thearteries were pressurized to 50 mmHg at 37° C. and were allowed toequilibrate for 45 minutes before experimentation. After submaximalpreconstriction with phenylephrine (2 μM), increases in luminal diameterin response to acetylcholine (ACh: 1×10⁻⁹-1×10⁻⁴ M; Sigma-Aldrich Corp.)with and without co-administration of the NO synthase inhibitor, L-NAME,0.1 mM, 30 minute incubation; (Sigma-Aldrich Corp.) or the superoxidedismutase mimetic 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl(TEMPOL, 0.1 mM, 1 hour incubation; Sigma-Aldrich Corp.) weredetermined. Endothelium-independent dilation was determined byvasodilation in response to the NO donor sodium nitroprusside (SNP:1×10⁻¹⁹-1×10⁻⁴ M; Sigma-Aldrich Corp.).

All dose-response data are presented as percent dilation, i.e. FIGS. 1A,1C and 2A. Preconstriction was calculated as a percentage of maximalintra-luminal diameter according to the following formula:Preconstriction (%)=(D _(m) −D _(b))/D _(m)×100Because of differences in maximal carotid artery diameter between youngand old animals, vasodilator responses were recorded as actual diametersexpressed as a percentage of maximal response according to the followingformula:Dilation (%)=(D _(s) −D _(b))/(D _(m) −D _(b))×100Where D_(m) is maximal intra-luminal diameter at 50 mmHg, D_(b) is thesteady-state intra-luminal diameter following preconstriction before thefirst addition of a drug, and D_(s) is the steady-state intra-luminaldiameter recorded after the addition of a drug.

NO-dependent dilation was determined from maximal EDD (i.e., dilationwith the highest dose [1×10⁻⁴ M] ACh) in the absence or presence ofL-NAME according to the following formula:NO-dependent dilation (%)=Maximum dilation_(ACh)−Maximaldilation_(ACh+L-NAME)

In Vivo Aortic Pulse Wave Velocity.

Aortic pulse wave velocity (aPWV) was measured as described previously(Sindler A. L. et al. 2011, Aging Cell. 10, 429-437; Fleenor B. S. etal. 2012b, Exp Gerontol. 47, 588-594). Briefly, mice were anesthetizedwith 2% isoflurane and placed supine on a heating board with legssecured to electrocardiogram (ECG) electrodes. Aortic blood flowvelocity was measured with two Doppler probes placed at the transverseaortic arch and abdominal aorta, respectively. Pre-ejection time, thetime between the R-wave of the ECG to foot of the Doppler signal, wasdetermined for each site. aPWV was calculated by dividing the distancebetween the transverse and abdominal probes by the difference in thethoracic and abdominal pre-ejection times.

In Vitro Elastic Modulus.

In vitro intrinsic mechanical properties of the thoracic aorta weredetermined and used to calculate the elastic modulus as previouslydescribed. Aortic segments (˜1.5 mm in length) from mice were cleaned ofperivascular fat and other surrounding tissue. They were then loadedonto a calibrated, pre-heated (37° C.) wire myograph chamber (DMT Inc.)containing calcium-free phosphate buffered saline. Aortic samples werepre-stretched for 3 minutes to a 1-mm luminal diameter displacement thatwas returned to the non-stretched baseline, and this was repeated twice.To begin the experiment after pre-stretching, segments were stretched toa baseline force of 1 mN. Luminal displacement was increasedincrementally (˜10% increase) every 3 minutes, and the force wasrecorded following every 3-minute time period. Displacement wasincreased until mechanical failure of the tissue occurred, defined by anobserved transient decrease in force. Stress and strain were calculatedwhere stress was defined as: t=λL/2HD. t=one-dimensional stress,λ=strain, L=one-dimensional load applied, H=wall thickness, D=length ofvessel. Strain was defined as: λ=Ad/d(i). λ=strain, Ad=change indiameter, d(i)=initial diameter. The slope of the stress-strain curvewas used to determine the elastic modulus as previously described(Fleenor B. S. et al. 2012b, Exp Gerontol. 47, 588-594).

Aortic Superoxide Production.

Measurement of superoxide production in the thoracic aorta was performedusing electron paramagnetic resonance (EPR) spectroscopy, as previouslydescribed (LaRocca T. J. et al. 2013, Mech Ageing Dev. 134, 314-320).The aorta was removed and dissected free of perivascular fat and othersurrounding tissue. 1-mm aortic segments were incubated for 1 hour at37° C. in Krebs-Hepes buffer with the superoxide-specific spin probe1-hydroxy-3methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH; 0.5 mM;Enzo Life Sciences, Inc., Farmington, N.Y., USA) for detection ofwhole-cell superoxide production. The signal amplitude was analyzedusing an MS300 X-band EPR spectrometer (Magnettech GmbH, Berlin,Germany) with the following settings: centrefield, 3350 G; sweep, 80 G;microwave modulation, 3000 mG, and microwave attenuation, 7 dB. Data arepresented relative to the YC group mean.

Western Blotting.

Aortas were used as a surrogate large elastic artery to providesufficient tissue for analysis of protein expression by Western blot asdescribed previously (Donato A. J. et al. 2013, J Physiol. 589,4545-4554; Gano L. B. et al. 2014, Am J Physiol Heart Circ Physiol. 307,H1754-1763). Aortas were excised, cleared of perivascular fat and othersurrounding tissues, and frozen in liquid nitrogen before storage at−80° C. The tissue was homogenized in ice-cold RadioimmunoprecipitationAssay (RIPA) lysis buffer containing protease and phosphatase inhibitors[Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, Ind., USA) and0.01% phosphatase inhibitor cocktail (Sigma Aldrich Corp.)] andpulverized using a Bullet Blender. Protein was loaded on 4-12%polyacrylamide gels (12 μg per well), separated by electrophoresis, andtransferred onto nitrocellulose membranes (Criterion System; Bio-RadLaboratories, Inc., Hercules, Calif., USA) for Western blot analysis.Membranes were incubated with the following primary antibodies overnightat 4° C.: anti-nitrotyrosine (NT 1:500; Abcam, Cambridge, Mass., USA),anti-sirtuinl (SIRT1 1:1000; Abcam), anti-p65 (subunit of nuclear factorkappa B (NFκB; 1:500 Cell Signaling Technology Inc., Danvers, Mass.,USA)), and anti-acetylated p65 (subunit of nuclear factor kappa B[ac-NFκB; 1:500 Cell Signaling Technology Inc.]). Proteins werevisualized on a digital acquisition system (ChemiDoc-It; UVP, Inc.,Upland, Calif., USA) using chemiluminescence with horseradishperoxidase-conjugated secondary antibodies (Jackson ImmunoResearchLaboratories, Inc., Westgrove, Pa., USA), enhanced chemiluminescence(ECL) substrate (Pierce Biotechnology, Inc., Rockford, Ill., USA).Relative intensity was quantified using Image-J Software Version 1.0.All data were normalized to expression of alpha smooth muscle actin (aactin 1:5000; Abcam). The ratio of acetylated to total p65 wasdetermined by running two identical Western blots. One membrane wasprobed for acetylated p65, while the other was probed for total p65.Each value was normalized to a actin in the corresponding gel. The ratioof acetylated to total p65 was determined for each animal and thennormalized to the YC group mean.

Immunohistochemistry.

Immunohistochemistry was used to determine aortic expression of collagentype I and elastin as previously described (Fleenor B. S. et al. 2010 JPhysiol. 588, 3971-3982). Thoracic aorta segments were excised andcleared of perivascular fat and other surrounding tissue. The segmentswere frozen in optimal cutting temperature compound (OCT; FisherScientific Inc., Waltham, Mass., USA) in liquid nitrogen-cooledisopentane. Aortic segments (7 μm each) were fixed in acetone for 10minutes and washed in Tris Buffer. All slides were stained with the DakoEnVision™+ System-HRP-DAB-kit (Dako, Carpinteria, Calif.) according tothe manufacturer's protocol using the following primary antibodies:collagen type I (Col1; 1:100, Millipore Corp., Temecula, Calif., USA)and alpha elastin (a elastin; 1:50, Abcam). Primary antibodies wereincubated for 1 hour at 4° C. The labeled polymer was applied for 30minutes and staining was visualized after a 4-minute exposure todiaminobenzidine (DAB). Slides were then dehydrated and coverslipped.Digital photomicrographs were obtained using a Nikon Eclipse TS100photomicroscope, and quantification was performed with Image-J SoftwareVersion 1.0. The adventitial and medial layers for each sample werequantified together for whole vessel expression. Slides from multiplebatches were normalized to the same representative YC animal andnormalized to the YC mean of each staining day.

Statistical Analyses.

Data are presented as mean±SEM in text, figures and tables. All analyseswere performed with SPSS. A one-way ANOVA (Analysis of Variance) wasused to analyze morphological characteristics, maximum EDD, NO-mediatedEDD, EPR spectroscopy, aPWV, elastic modulus, Western blots, andimmunohistochemistry. Within-group differences in the maximal EDD doseresponse to acetylcholine in the absence vs. presence of TEMPOL wasdetermined using two-factor (group×treatment) repeated-measures ANOVA.When a significant main effect was observed, Tukey post-hoc tests wereused to determine specific pairwise differences. Significance was set atp<0.05.

Results

Animal Characteristics and NMN Intake

Selected morphological characteristics are shown in Table 1.

TABLE 1 YC OC YNMN ONMN Body mass (g) 30.1 ± 0.7 30.3 ± 0.6  28.0 ± 0.930.8 ± 0.6  Heart mass (mg) 145 ± 6  176 ± 7 * 139 ± 7  182 ± 8 * Quadmass (mg) 176 ± 5  151 ± 4 * 185 ± 6  146 ± 6 * Gastroc mass (mg) 151 ±3  124 ± 3 * 148 ± 8  126 ± 2 * WAT mass (mg) 730 ± 65  472 ± 41 * 600 ±35  430 ± 50 * SubQ fat mass (mg) 334 ± 32  207 ± 17 * 252 ± 16  201 ±23 * Values are mean ± SEM * p < 0.05 vs. YC. n = 13-19/group. Quad,quadriceps; Gastroc, gastrocnemius; WAT, white adipose tissue; SubQ,subcutaneous fat.

There were no significant differences in body mass across the fourgroups. An age-related increase in heart mass and a decrease in fat andmuscle mass, which were not altered with NMN treatment, were observed.All animals consumed the same quantity of food throughout the durationof the study, and NMN intake was similar in both young and old treatedgroups.

NMN Treatment Restores Maximum EDD and NO-Mediated EDD in Old Mice

Baseline carotid artery diameters (μm) assessed ex vivo were greater inold mice (controls: 480±17; treated: 478±9) vs. young mice (controls:424±4; treated: 432±5). NMN treatment had no significant effect onbaseline carotid artery diameters. Maximum EDD to acetylcholine assessedex vivo was lower in old control compared with young control mice andwas mediated in part by a diminished nitric oxide (NO) dilatoryinfluence, as indicated by a smaller reduction in EDD in the presencevs. absence of the NO synthase inhibitor N-G-nitro-L-arginine (L-NAME)(FIGS. 1A, 1B, n=13-22/group). NMN treatment rescued EDD in old mice byrestoring NO-mediated dilation, but had no effect in young treatedanimals (FIGS. 1A, 1B). There were no significant differences in maximalEDD or NO-mediated EDD in young and old treated animals vs. youngcontrols. Endothelium-independent dilation to the NO donor sodiumnitroprusside, a control to assess vascular smooth muscle sensitivity toNO, was not significantly different among the groups (FIG. 1C,n=7-22/group). Values are mean±SEM. *p<0.05 vs. all.

NMN Reduces Vascular Oxidative Stress

Ex vivo incubation with the superoxide dismutase mimetic,4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), restored EDD incarotid arteries of old control animals, while having no significanteffect in the other groups (FIG. 2A, n=5-9/group. * p<0.05 vs. TEMPOL),indicating excessive superoxide-mediated endothelial dysfunction withaging. To further assess the influence of aging and NMN treatment onoxidative stress in arteries, aortas were used because of the greateramount of tissue provided for biochemical assay. Compared with youngmice, aortas from old animals exhibited increased superoxide production,directly assessed by electron paramagnetic resonance spectroscopy (FIG.2B; values are normalized to YC mean value, representative EPR signalbelow, n=4-9/group), and increased nitrotyrosine abundance (FIG. 2C), amarker of oxidative protein modification and cellular footprint ofoxidative stress. NMN treatment ameliorated the age-related increase insuperoxide production (FIG. 2B) and markedly reduced aorticnitrotyrosine abundance in old mice (FIG. 2C, n=4-6/group), while havingno significant effect in young mice. Data are expressed relative toα-smooth muscle actin and normalized to YC mean value. Values aremean±SEM. * p<0.05 vs. all; # p<0.05 vs. YC. Together, these dataindicate that NMN reverses age-associated oxidative stress in arteries,which, in turn, mediates improvements in endothelial function.

NMN Treatment Normalizes Aortic Stiffness in Old Mice

Large elastic artery stiffness, as assessed in vivo by aPWV, was greaterin old control compared to young control mice (FIG. 3A, n=5-9/group).NMN treatment reversed the age-associated increase in aPWV in old mice,while having no significant effect in young mice (FIG. 3A). Similarly,the elastic modulus, an in vitro index of intrinsic arterial stiffness,was higher in old controls compared to young and was normalized with NMNtreatment (FIG. 3B, n=5-6/group). Thoracic aortas from old controlanimals exhibited markedly increased collagen type I expression (FIG.3C, n=4-9/group) and diminished elastin (FIG. 3D) compared to youngcontrols. In old mice, NMN reduced arterial collagen type I to levels ofyoung mice (FIG. 3C), and increased elastin to levels not significantlydifferent from young mice (FIG. 3D, n=4-11/group). Values normalized toYC mean value. Values are mean±SEM. Bars=100 μm. * p<0.05 vs. all; #p<0.05 vs. YC.

NMN Treatment Activates SIRT1

Mean levels of aortic SIRT1 expression were ˜50% lower in the oldanimals, compared to young mice, although the difference did not reachstatistical significance (FIG. 4A, n=5-7/group). NMN treatment increasedSIRT1 protein expression in young animals and tended to increase SIRT1in old animals (FIG. 4A). SIRT1 activation was determined by assessingthe ratio of acetylated to total NFκB (p65 subunit) (FIG. 4B). Thisratio was markedly higher in aorta of old control animals compared toyoung controls (p<0.05), indicating that aortic SIRT1 activity wasreduced with aging. Data are expressed relative to α-smooth muscle actinand normalized to YC mean value. Values are mean±SEM, # p<0.05 vs. YC; †p<0.05 vs. OC. NMN treatment restored aortic SIRT1 activity in oldanimals (p<0.05; FIG. 4B, n=5-11/group).

The data demonstrates that 8 weeks of NMN treatment restored arterialSIRT1 activity and ameliorated age-associated endothelial dysfunctionand large elastic artery stiffening in male C57Bl/6 mice. Theseimprovements were associated with restored NO bioavailability, reducedoxidative stress, and complete or partial normalization of structuralproteins in the arterial wall.

NMN Improves NO-Mediated EDD and Reduces Arterial Oxidative Stress

Endothelial dysfunction is the major antecedent to atherosclerosis, apredictor of clinical CVD risk, and is linked to many common disordersof aging including cognitive impairments, Alzheimer's Disease, motordysfunction, insulin resistance and sarcopenia (Heitzer T. et al. 2001,Circulation. 104, 2673-2678; Gokce N. et al. 2002, Circulation. 105,1567-1572; Widlansky M. E. et al. 2003, J Am Col1 Cardiol. 42,1149-1160; Seals D. R. et al. 2011, Clin Sci 120, 357-375; Seals D. R.et al. 2014, Physiology (Bethesda). 29, 250-264). Motor and cognitivefunctions are associated with the maintenance of independence andquality of life in older adults and represent clinical endpoints. Areversal of age-associated endothelial dysfunction by oral NMN treatmentwas demonstrated. Aging in the control animals was associated with animpairment in ex vivo carotid artery EDD in response to acetylcholinedue to reduced NO-mediated dilation (FIGS. 1A-1B). The latter wasdetermined as the difference in EDD in the absence vs. presence of NOproduction achieved by co-administration of the NO synthase inhibitor,NG-nitro-L-arginine methyl ester (L-NAME) (Max Dilation_(ACh)−MaxDilation_(ACh+L-NAME), n=5-12/group). NMN treatment substantially orcompletely restored EDD by restoring NO-mediated dilation. Theimprovement in EDD with NMN treatment in old mice is not believed to bedue to an increase in vascular smooth muscle sensitivity to NO becauseNMN did not influence dilation in response to administration of a NOdonor (sodium nitroprusside), i.e., a dilation that is not induced byendothelial NO production.

The results demonstrate that the effects of aging and NMN treatment onendothelial function were mediated by differences in oxidative stress.Pharmaco-functional experiments revealed that arteries from olduntreated mice showed restoration of maximal EDD in the presence of thesuperoxide dismutase mimetic, TEMPOL, indicating that excessivesuperoxide-related oxidative stress may be the cause of theage-associated impairment of EDD (FIG. 2A). In contrast to the untreatedold mice, TEMPOL caused no significant improvement in EDD in old micereceiving NMN treatment. NMN treatment may restore EDD in the oldanimals by abolishing superoxide-mediated suppression of endothelialfunction with aging.

These function-based observations are supported by direct assessments ofarterial superoxide production using electron paramagnetic resonancespectroscopy, as well as results for nitrotyrosine, a cellular marker ofoxidative stress. Aging in control animals was associated with markedincreases in both aortic superoxide production and nitrotyrosineabundance (FIGS. 2B-2C). In response to NMN treatment, a normalizationof aortic superoxide production and a marked reduction in nitrotyrosineabundance in old mice was observed.

Collectively, these findings support the various aspects and embodimentsof the invention comprising prevention of age-related decline,prevention of stress with treatment of NMN, that oxidative stress is akey contributor to age-associated endothelial dysfunction and that thesuppression of oxidative stress may be a major mechanism by which NMNexerts its beneficial effects on endothelial function in old animals.

NMN Reduces Large Elastic Artery Stiffness

NMN treatment reverses the age-associated increase in two functionalindices of aortic stiffness: aPWV, the gold standard clinical measure oflarge elastic artery stiffness, and the elastic modulus, an in vitromeasure of the intrinsic mechanical properties of arteries (FIGS.3A-3B).

NMN treatment reverses the accumulation of whole-vessel collagen type Iand enhances arterial elastin in old mice (FIGS. 3C-3D). NMN reducesarterial stiffness, at least in part, by ameliorating the structuralchanges that occur to arteries with advancing age.

Reductions in aortic collagen in old mice subjected to other short-termlate-life behavioral or pharmacological interventions has been observed.NMN also induced a partial restoration of aortic elastin to levels notsignificantly different from young control animals. An increase inaortic elastin with any other late-life lifestyle or pharmacologicalintervention in mice has not been observed, although lifelong CRprotects against the loss of elastic properties within the arteries,including elastin degradation (Fornieri C. et al. 1999, Connect TissueRes. 40, 131-143). These results demonstrate that NMN partially restoresarterial elastin.

Activation of SIRT1 by NMN

NAD⁺ measurements in the aortic samples could not be acquired withconfidence, likely due to the limited amount of tissue and the timeneeded to extract the aorta compared with larger, more easily obtainedtissues such as skeletal muscle or white adipose tissue. Treatment withNMN selectively restored the activity of SIRT1 in the arteries of oldmice to that of young controls, as indicated by a decrease in the ratioof acetylated to total p65 subunit of the transcription factor NFκB(FIG. 4B).

All references cited herein are hereby incorporated by reference intheir entirety.

It is believed that the disclosure set forth above encompasses at leastone distinct invention with independent utility. While the invention hasbeen disclosed in the exemplary forms, the specific embodiments thereofas disclosed and illustrated herein are not to be considered in alimiting sense as numerous variations are possible. Equivalent changes,modifications and variations of various embodiments, materials,compositions and methods may be made within the scope of the presentinvention, with substantially similar results. The subject matter of theinventions includes all novel and non-obvious combinations andsubcombinations of the various elements, features, functions and/orproperties disclosed herein.

Benefits, other advantages, and solutions to problems have beendescribed herein with regard to specific embodiments. However, thebenefits, advantages, solutions to problems, and any element orcombination of elements that may cause any benefit, advantage, orsolution to occur or become more pronounced are not to be construed ascritical, required, or essential features or elements of any or all theclaims of the invention. Many changes and modifications within the scopeof the instant invention includes all such modifications. Correspondingstructures, materials, acts, and equivalents of all elements in theclaims below are intended to include any structure, material, or actsperforming the functions in combination with other claim elements asspecifically claimed. The scope of the invention should be determined bythe appended claims and their legal equivalents, rather than by theexamples given above.

The invention claimed is:
 1. A method for treatment of vascularendothelial dysfunction, comprising: determining an indicator ofvascular endothelial dysfunction in a subject, wherein the indicator isselected from a group consisting of: endothelium-dependent dilation;artery stiffness; and aortic pulse wave velocity; and administering adaily dose of a composition comprising nicotinamide mononucleotide and apharmaceutical excipient, wherein the composition comprises from about 1mg to about 25 mg of nicotinamide mononucleotide per kg body weight perday and wherein the composition is administered chronically to saidsubject in response to the indicator.
 2. The method of claim 1, furthercomprising determining a subsequent effect on vascular endothelialdysfunction in the subject.
 3. The method of claim 1, wherein theendothelium-dependent dilation is associated with increased superoxideproduction.
 4. The method of claim 1, wherein the endothelium-dependentdilation is associated with decreased SIRT1 expression.
 5. The method ofclaim 1, wherein extent of endothelium-dependent dilation and/or arterystiffness is decreased in response to administration of the compositioncomprising nicotinamide mononucleotide.
 6. The method of claim 5,wherein the decrease in the extent of endothelium-dependent dilationfurther comprises a decrease in superoxide production and an increase innitric oxide bioavailability.
 7. The method of claim 5, wherein thedecrease in the extent of endothelium-dependent dilation furthercomprises an increase in SIRT1 protein expression and activity.
 8. Themethod of claim 1, wherein the composition is administered over a periodof time of about 30 days, about 3 months, about 6 months, about 12months, about 18 months, about 2 years, about 5 years, about 7 years,about 10 years, about 15 years, about 20 years, about 25 years, about 30years, about 35 years, about 40 years, or continued therapy over thelifetime of the subject.
 9. The method of claim 1, wherein thecomposition comprises about 18 mg of nicotinamide mononucleotide per kgbody weight per day.
 10. A method of decreasing the extent ofendothelium-dependent dilation and/or arterial stiffness in a subject,comprising: determining the extent of endothelium-dependent dilationand/or arterial stiffness in a subject, wherein the indicator isselected from a group consisting of: endothelium-dependent dilation;artery stiffness; and aortic pulse wave velocity; and administering adaily dose of a composition comprising nicotinamide mononucleotide and apharmaceutical excipient wherein the composition comprises from about 1mg to about 25 mg of nicotinamide mononucleotide per kg body weight perday and wherein the composition is administered chronically to saidsubject.
 11. The method of claim 10, wherein a decrease in the extent ofendothelium-dependent dilation and/or arterial stiffness is associatedwith an increase in bioavailability of nicotinamide adenine dinucleotide(NAD+).
 12. The method of claim 11, wherein a decrease in the extent ofendothelium-dependent dilation and/or arterial stiffness furthercomprises a decrease in superoxide production, an increase inbioavailability of nitric oxide, and/or an increase in SIRT1 proteinexpression and activity.
 13. The method of claim 10, wherein thecomposition comprises about 18 mg of nicotinamide mononucleotide per kgbody weight per day.
 14. A method for modulating endothelium-dependentdilation and/or arterial stiffness in a subject, comprising:administering a daily dose of a composition comprising nicotinamidemononucleotide and a pharmaceutical excipient wherein the compositioncomprises from about 1 mg to about 25 mg of nicotinamide mononucleotideper kg body weight per day and wherein the composition is administeredchronically to said subject.
 15. The method of claim 14, wherein theextent of endothelium-dependent dilation is associated with increasedsuperoxide production.
 16. The method of claim 14, wherein the extent ofendothelium-dependent dilation is associated with decreased SIRT1expression.
 17. The method of claim 14, wherein the extent ofendothelium-dependent dilation and/or artery stiffness is decreased inresponse to administration of the composition comprising nicotinamidemononucleotide.
 18. The method of claim 14, wherein the extent ofendothelium-dependent dilation further comprises a decrease insuperoxide production and an increase in nitric oxide bioavailability.19. The method of claim 14, wherein the extent of endothelium-dependentdilation further comprises an increase in SIRT1 protein expression andactivity.
 20. The method of claim 14, wherein the composition isadministered over a period of time of about 30 days, about 3 months,about 6 months, about 12 months, about 18 months, about 2 years, about 5years, about 7 years, about 10 years, about 15 years, about 20 years,about 25 years, about 30 years, about 35 years, about 40 years, orcontinued therapy over the lifetime of the subject.
 21. The method ofclaim 14, wherein the composition comprises about 18 mg of nicotinamidemononucleotide per kg body weight per day.